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A Novel Method For Tissue Glutathione Redox Couple Analysis Using HPLC‐Boron Doped Diamond Detection (BDD)
Author(s) -
Park Hea Jin,
Courtemanche Kevin,
Bruno Richard S.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.729.3
Subject(s) - glutathione , chemistry , glutathione disulfide , high performance liquid chromatography , perchloric acid , chromatography , acetonitrile , quantitative analysis (chemistry) , detection limit , redox , biochemistry , inorganic chemistry , enzyme
Glutathione (GSH/GSSG) is a marker of oxidative stress. HPLC‐FL and ‐UV approaches have been used, but are challenging, time‐consuming, and column life is limited. Electrochemical (ECD) approaches have been used, but the high potential required for disulfide oxidation results in signal degradation. Thus, we aimed to measure hepatic GSH and GSSG simultaneously using HPLC‐BDD detection, a novel ECD approach having greater stability at high electrochemical potentials. Mice (n = 4/group) were injected ip with lipopolysaccaride (LPS, 0 mg or 4 mg/kg BW) and sacrificed at 24 h. Liver homogenates were mixed with perchloric acid and the isolated supernatant was used for analysis. The sample was separated isocratically at 32°C on C18 column using sodium dihydrogen phosphate, octanesulfonic acid, and acetonitrile as the mobile phase. Hydrodynamic voltamogram experiments indicated an optimal applied potential of 1400 mV for GSH and GSSG. GSH and GSSG retention times were 6.6 ± 0.02 and 16.6 ± 0.12 min, respectively, and standards were linear (r > 0.99) between 1‐8000 pmol injected for each. Within‐ and between‐day coefficient of variations were <2% and <7%, respectively. LPS decreased the GSH/GSSG ratio, reflective of decreased GSH and increased GSSG. These data indicated that hepatic glutathione can be measured reliably with minimal sample preparation and greater sample throughput using HPLC‐BDD.